C1-INH compositions and methods for the prevention and treatment of disorders associated with C1 esterase inhibitor deficiency

ABSTRACT

Compositions and methods for the treatment and/or prevention of disorders associated with C1 esterase inhibitor deficiency are disclosed.

This application is a continuation of PCT/US2014/030309, filed Mar. 17,2014, which claims priority to U.S. Provisional Patent Application No.61/791,399, filed Mar. 15, 2013, the disclosure of which is herebyincorporated by reference.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 14, 2015, isnamed 2006685-1184_SL.txt and is 4,606 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic agents andmethods of use thereof. Specifically, the instant invention providescompositions and methods for the treatment and/or prevention ofdisorders associated with C1 esterase inhibitor deficiency.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Full citations of these references can be foundthroughout the specification. Each of these citations is incorporatedherein by reference as though set forth in full.

Hereditary angioedema (HAE) is a rare, life-threatening, geneticdisorder caused by a deficiency of the C1 esterase inhibitor (seegenerally www.haei.org and www.haea.org). At least 6,500 people in theUnited States and at least 10,000 people in Europe have HAE. HAEpatients experience recurrent, unpredictable, debilitating,life-threatening attacks of inflammation and submucosa/subcutaneousswelling. The inflammation is typically of the larynx, abdomen, face,extremities, and urogenital tract. This genetic disorder is a result ofa defect in the gene controlling the synthesis of the C1 esteraseinhibitor. Accordingly, restoring the levels of active C1 esteraseinhibitor in these patients to or near normal levels is an effectivemeasure for treating HAE. Still, new and improved methods of treatingand preventing disorders associated with a deficiency of the C1 esteraseinhibitor, such as HAE, are desired.

SUMMARY OF THE INVENTION

In accordance with the instant invention, methods for inhibiting,treating, and/or preventing a disorder associated with a deficiency inC1 esterase inhibitor in a subject are provided. In a particularembodiment, the method comprises administering a composition comprisingat least one C1 esterase inhibitor.

In accordance with the instant invention, therapeutic compositions arealso provided. In a particular embodiment, the composition comprises atleast one C1 esterase inhibitor and, optionally, at least onepharmaceutically acceptable carrier for delivery (e.g. intravenous orsubcutaneous delivery). Kits comprising a composition comprising atleast one C1 esterase inhibitor are also provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an amino acid sequence of human C1 esterase inhibitor.

FIG. 2 provides a graph of the effect of protein concentration onviscosity for initial spin concentration samples.

DETAILED DESCRIPTION OF THE INVENTION

The restoration of active C1 esterase inhibitor levels in patientshaving a disorder associated with deficient or reduced levels of activeC1 esterase inhibitor (e.g., HAE) is an effective measure for treatingsuch disorders. Currently, C1 esterase inhibitor (such as Cinryze®(ViroPharma, Inc.; Exton, Pa.)) is administered to a patientintravenously by a medical professional. Herein, formulations of a C1esterase inhibitor (such as Cinryze®) are provided which are alsoeffective for subcutaneous (SC) administration. Surprisingly, thesubcutaneous administration of the C1 esterase inhibitor is sufficientto maintain the blood levels of the C1 esterase inhibitor. The SCadministration of a C1 esterase inhibitor fulfills an unmet medical needdue to the limitations of intravenous administration in HAE patients.

In accordance with the instant invention, compositions and methods forinhibiting (e.g., reducing or slowing), treating, and/or preventing adisorder associated with C1 esterase inhibitor deficiency in a subjectare provided. In a particular embodiment, the methods compriseadministering (e.g., subcutaneously or intravenously) to a subject inneed thereof at least one C1 esterase inhibitor. In a particularembodiment, the C1 esterase inhibitor is administered subcutaneouslyafter an initial administration of the C1 esterase inhibitorintravenously.

C1 esterase inhibitors are also known as C1 inhibitors (C1 INH). C1esterase inhibitors are inhibitors of complement C1 and belong to thesuperfamily of serine proteinase inhibitors. Human C1 esterase inhibitoris a protein of 500 amino acids, including a 22 amino acid signalsequence (Carter et al. (1988) Eur. J. Biochem., 173:163). In plasma,the C1 esterase inhibitor is a heavily glycosylated glycoprotein ofapproximately 76 kDa (Perkins et al. (1990) J. Mol. Biol., 214:751). Theactivity of a C1 esterase inhibitor may be assayed by known methods(see, e.g., Drouet et al. (1988) Clin. Chim. Acta., 174:121-30). In aparticular embodiment, the C1 esterase inhibitor is human. An amino acidsequence of human C1 esterase inhibitor is provided in GenBank AccessionNo. CAA30314 (see also GeneID: 710, which also provides nucleotidesequences of the C1 esterase inhibitor) and FIG. 1. A C1 esteraseinhibitor for use in the methods of the instant invention may have anamino acid sequence that has at least 65, 70, 75, 80, 85, 90, 95, 98,99, or 100% identity with the amino acid sequence of FIG. 1. The C1esterase inhibitor may be isolated or purified from plasma (e.g., humanplasma) or recombinantly produced. When purified from plasma, the C1esterase inhibitor may be nanofiltered and pasteurized. In a particularembodiment, the plasma-derived C1 esterase inhibitor is Cinryze®. In aparticular embodiment, the C1 esterase inhibitor is present in thecompositions of the instant invention at high concentration. Indeed,compositions comprising very high levels of C1 esterase inhibitor havebeen determined to be surprisingly stable and active. In a particularembodiment, the C1 esterase inhibitor is present at about 250 U/ml toabout 1000 U/ml, about 400 U/ml to about 600 U/ml, or about 500 U/ml.

In a particular embodiment, the compositions of the instant invention donot contain citrate or citric acid. The compositions lacking citrate andcitric acid are particularly useful for the subcutaneous administrationof the C1 esterase inhibitor as citrate/citric acid can cause aninjection site reaction. In a particular embodiment, the buffer of theinstant compositions is sodium phosphate (e.g., about 5 mM to about 50mM sodium phosphate, about 10 mM to about 30 mM sodium phosphate, orabout 20 mM sodium phosphate). In a particular embodiment (e.g., forintravenous administration), the buffer of the instant compositionscomprises a carboxylic group. For example, the buffer may be, withoutlimitation, citrate, succinate, tartarate, maleate, acetate, and saltsthereof. In a particular embodiment, the buffer of the instantcomposition is citrate or sodium citrate (e.g., about 5 mM to about 50mM sodium citrate, about 10 mM to about 30 mM sodium citrate, or about20 mM sodium citrate).

The compositions of the instant invention may have a pH range of about6.5 or higher, particularly about 6.5 to about 8.0, particularly about6.5 to about 7.5, and more particularly about 6.5 to about 7.0.

The compositions of the instant invention may also comprise polysorbate80 (TWEEN). Compositions comprising polysorbate 80 are particularlyuseful as they reduce/mitigate protein aggregation. Polysorbate 80 canalso limit protein interactions when the composition comes into contactwith silicon containing lubricants/oils such as those used in syringesand other administration devices. Compositions comprising polysorbate 80are also useful for lyophilized preparations. In a particularembodiment, the polysorbate 80 is present at a concentration of about0.01% to about 0.1%, particularly about 0.025% to about 0.075%,particularly about 0.05%.

The compositions of the instant invention may also comprise sucrose.Sucrose can be added as a “bulking” agent as well as a lyo-protectant.In a particular embodiment, sucrose is added to compositions to belyophilized. In a particular embodiment, the compositions comprise about25 mM to about 125 mM sucrose, particularly about 50 mM to about 100 mMsucrose.

The compositions of the instant invention may also comprise at least oneamino acid or salt thereof, particularly methionine and/or arginine.Arginine carries a positive charge on its side chain can be used tobuffer solutions with phosphate. Methionine acts as a stabilizer (e.g.,by limiting oxidation). The amino acids may be present in thecomposition as individual amino acids or present as short peptides(e.g., 2 to about 5 amino acids, particularly di-peptides ortri-peptides).

As stated hereinabove, the instant invention encompasses methods oftreating, inhibiting, and or preventing any condition or diseaseassociated with an absolute or relative deficiency of functional C1esterase inhibitor. Such disorders include, without limitation, acquiredangioedema (AAE) and hereditary angioedema (HAE). In a particularembodiment, the disorder is HAE and/or the attacks associated therewith.As stated hereinabove, HAE is a life-threatening and debilitatingdisease that manifests as recurrent, submucosal/subcutaneous swellingattacks due to a deficiency of C1 esterase inhibitor (Zuraw, B. L.(2008) N. Engl. J. Med., 359:1027-1036). In a particular embodiment, thehereditary angioedema is type I or type II. Both type I and type II havea defective gene for the synthesis of C1 esterase inhibitor that produceeither no C1 inhibitor (HAE type I) or a dysfunctional C1 inhibitor (HAEtype II) (Rosen et al. (1965) Science 148: 957-958; Bissler et al.(1997) Proc. Assoc. Am. Physicians 109: 164-173; Zuraw et al. (2000) J.Allergy Clin. Immunol. 105: 541-546; Bowen et al. (2001) Clin. Immunol.98: 157-163).

The methods of the instant invention encompass the administration of atleast one C1 esterase inhibitor. Compositions comprising at least one C1esterase inhibitor and, optionally, at least one pharmaceuticallyacceptable carrier (e.g., one suitable for subcutaneous or intravenousadministration) are encompassed by the instant invention. Suchcompositions may be administered, in a therapeutically effective amount,to a patient in need thereof for the treatment of a disorder associatedwith C1 esterase inhibitor deficiency. The instant invention alsoencompasses kits comprising at least one composition of the instantinvention, e.g., a composition comprising at least one C1 esteraseinhibitor and, optionally, at least one pharmaceutically acceptablecarrier (e.g., one suitable for intravenous or subcutaneousadministration). The kits may further comprise at least one ofreconstitution buffer(s), syringes (e.g., disposable) for parenteral(e.g., subcutaneous) injection, and instruction material. In aparticular embodiment, the kit comprises at least one pre-loaded syringecomprising the C1 esterase inhibitor and at least one pharmaceuticallyacceptable carrier. For example, a syringe may be loaded with at leastone C1 esterase inhibitor with at least one pharmaceutically acceptablecarrier for administration (e.g., intravenous or subcutaneousadministration). Alternatively, a single syringe may be loaded withlyophilized C1 esterase inhibitor. In a particular embodiment, thepreloaded syringes have a pharmaceutical composition that containspolysorbate 80 as a component (e.g., in an amount that preventsprotein-silicone interaction or protein aggregation).

The agents and compositions of the present invention can be administeredby any suitable route, for example, by injection (e.g., for local(direct) or systemic administration. In a particular embodiment, thecomposition is administered subcutaneously or intravenously. In general,the pharmaceutically acceptable carrier of the composition is selectedfrom the group of diluents, preservatives, solubilizers, emulsifiers,adjuvants and/or carriers. The compositions can include diluents ofvarious buffer content (e.g., Tris HCl, acetate, phosphate), pH andionic strength; and additives such as detergents and solubilizing agents(e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid,sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol)and bulking substances (e.g., lactose, mannitol). The pharmaceuticalcomposition of the present invention can be prepared, for example, inliquid form, or can be in dried powder form (e.g., lyophilized for laterreconstitution).

In a particular embodiment, the compositions are formulated inlyophilized form. Where the compositions are provided in lyophilizedform, the compositions are reconstituted prior to use (e.g., within anhour, hours, or day or more of use) by an appropriate buffer (e.g.,sterile water, a sterile saline solution, or a sterile solutioncomprising the appropriate pharmaceutically acceptable carriers (e.g.,to reconstitute the compositions as described hereinabove). Thereconstitution buffer(s) may be provided in the kits of the instantinvention or may be obtained or provided separately.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media and the like which may be appropriate forthe desired route of administration of the pharmaceutical preparation,as exemplified in the preceding paragraph. The use of such media forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the molecules tobe administered, its use in the pharmaceutical preparation iscontemplated.

Selection of a suitable pharmaceutical preparation depends upon themethod of administration chosen. In this instance, a pharmaceuticalpreparation comprises the molecules dispersed in a medium that iscompatible with the tissue to which it is being administered. Methodsfor preparing parenterally or subcutaneously administrable compositionsare well known in the art (see, e.g., Remington's Pharmaceutical Science(E. W. Martin, Mack Publishing Co., Easton, Pa.)).

As stated hereinabove, agents of the instant invention are administeredparenterally—for example by intravenous injection into the blood streamand/or by subcutaneous injection. Pharmaceutical preparations forparenteral, intravenous, and subcutaneous injection are known in theart. If parenteral injection is selected as a method for administeringthe molecules, steps should be taken to ensure that sufficient amountsof the molecules reach their target cells to exert a biological effect.

Pharmaceutical compositions containing a compound of the presentinvention as the active ingredient in intimate admixture with apharmaceutical carrier can be prepared according to conventionalpharmaceutical compounding techniques. The carrier may take a widevariety of forms depending on the form of preparation desired foradministration, e.g., parenterally or subcutaneous. For parenterals, thecarrier will usually comprise sterile water, though other ingredients,for example, to aid solubility or for preservative purposes, may beincluded. Injectable suspensions may also be prepared, in which caseappropriate liquid carriers, suspending agents and the like may beemployed.

A pharmaceutical preparation of the invention may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unitof the pharmaceutical preparation appropriate for the patient undergoingtreatment. Each dosage should contain a quantity of active ingredientcalculated to produce the desired effect in association with theselected pharmaceutical carrier. Dosage units may be proportionatelyincreased or decreased based on the weight of the patient. Appropriateconcentrations for alleviation of a particular pathological conditionmay be determined by dosage concentration curve calculations.Appropriate dosage unit may also be determined by assessing the efficacyof the treatment.

The pharmaceutical preparation comprising the molecules of the instantinvention may be administered at appropriate intervals, for example,daily, every other day, every three days, five out of every 7 days, orat least one, two or three times a week or more until the pathologicalsymptoms are reduced or alleviated, after which the dosage may bereduced to a maintenance level. The appropriate interval in a particularcase would normally depend on the condition of the patient.

In a particular embodiment, the C1 esterase inhibitor is present in thecomposition or is administered in the range of about 100 Units to about10,000 Units; about 500 Units to about 5,000 Units; about 1,000 Units toabout 3,500 Units, or about 1,500 Units to about 2,500 Units. In aparticular embodiment, at least about 2,000 Units is used. In aparticular embodiment, a high initial dose of the C1 esterase inhibitor(as listed above (may be administered intravenously)) is used, followedby lower maintenance doses. For example, the high initial dose may be atleast 1.5, 2, 3, 4, or 5 times the subsequent doses. In a particularembodiment, the C1 esterase inhibitor is present in the maintenancecomposition or is administered for maintenance in the range of about 100Units to about 5,000 Units; about 250 Units to about 2,000 Units; about250 Units to about 1,000 Units; or about 500 Units. The high initialdoes of the C1 esterase inhibitor is optional in the methods of theinstantly claimed invention (e.g., may be optional with prophylacticmethods).

In a particular embodiment, the C1 esterase inhibitor is administeredwith a frequency and dosage so as to increase the C1 esterase inhibitorlevel to at least about 0.3 or, more particularly, 0.4 U/ml or more upto about 1 U/ml (1 Unit/ml is the mean quantity of C1 inhibitor presentin 1 ml of normal human plasma) in the blood of the subject. Forexample, the C1 esterase inhibitor level may be kept at or above 0.4U/ml for at least 50%, at least 75%, at least 90%, at least 95% or moreof time or all of the time (e.g., the time during which drug is beingadministered). For example, the administration of a 2000 U initial doseof C1 esterase inhibitor followed by 250 U everyday or 500 U every otherday results in the maintenance of just below 0.4 U/ml in blood. Further,the administration of a 2000 U initial dose of C1 esterase inhibitorfollowed by 1000 U every 3 days results in the maintenance of about 0.4U/ml in blood. Notably, for ease of use by the patient, less frequentadministrations may be preferred. The administration of a 2000 U initialdose of C1 esterase inhibitor followed by 500 U everyday with weekendholidays from administration (i.e., 5 out of 7 days) also results in themaintenance of about 0.4 U/ml or higher in blood. Notably, theadministration of only the maintenance doses leads to increased andphysiologically relevant blood levels of the C1 esterase inhibitor, butdelayed compared to those receiving an initial high dose.

DEFINITIONS

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

As used herein, the term “about” may refer to =5%, ±2%, or ±1%.

As used herein, the terms “host,” “subject,” and “patient” refer to anyanimal, including humans.

As used herein, the term “prevent” refers to the prophylactic treatmentof a subject who is at risk of developing a condition (e.g., HAE or HAEattack) resulting in a decrease in the probability that the subject willdevelop the condition.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disorder, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the condition, etc. In aparticular embodiment, the treatment of HAE results in at least areduction in the severity and/or number of HAE attacks.

The phrase “effective amount” refers to that amount of therapeutic agentthat results in an improvement in the patient's condition. A“therapeutically effective amount” of a compound or a pharmaceuticalcomposition refers to an amount effective to prevent, inhibit, treat, orlessen the symptoms of a particular disorder or disease.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative(e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid,sodium metabisulfite), solubilizer (e.g., TWEEN 80, Polysorbate 80),emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), water, aqueoussolutions, oils, bulking substance (e.g., lactose, mannitol),cryo-/lyo-protectants, tonicity modifier, excipient, auxilliary agent orvehicle with which an active agent of the present invention isadministered. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin (Mack PublishingCo., Easton, Pa.); Gennaro, A. R., Remington: The Science and Practiceof Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe,et al., Eds., Handbook of Pharmaceutical Excipients, AmericanPharmaceutical Association, Washington.

The term “isolated” may refer to protein, nucleic acid, compound, orcell that has been sufficiently separated from the environment withwhich it would naturally be associated (e.g., so as to exist in“substantially pure” form). “Isolated” does not necessarily mean theexclusion of artificial or synthetic mixtures with other compounds ormaterials, or the presence of impurities that do not interfere with thefundamental activity, and that may be present, for example, due toincomplete purification.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). In certain embodiments, the preparationcomprises at least 75% by weight, particularly 90-95% or more by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

The following example is provided to illustrate various embodiments ofthe present invention. The example is illustrative and is not intendedto limit the invention in any way.

EXAMPLE

Spin Concentration Studies

The protein was loaded into the spin concentrators and rotated at 10,500rpms for 5 to 10 minutes. When the samples stopped rotating, the finalvolumes in the spin concentrators were recorded and a rough proteinconcentration was calculated for each one. Additional protein was addedto the spin concentrators and rotated until the desired proteinconcentration was reached, at which point a UV measurement was made. Ateach target protein concentration a UV and viscosity measurement wasperformed. The above procedure continued until the viscosity of theprotein prevented the sample from being further concentrated.

Viscosity Measurements

Viscosity was determined by measuring the amount of time the sample tookto be drawn to a predetermined distance in a gel loading pipette tip. Inorder to calculate the sample viscosity, a standard curve was firstprepared using a set of standards with known viscosities. Sucrose (orBrix) solutions are suitable for preparing such a curve, but anymaterial with known viscosity at a defined temperature should beappropriate.

In order to make a measurement, the pipette plunger is depressed, thepipette tip is inserted into the sample vial, the plunger is released,and the time for the fluid to travel a predetermined distance in thepipette tip was measured with a stop watch. The distance used for theseexperiments was 30 μL of water. In important note, a pipette tip is onlyreliable for a single measurement, so multiple tips are used to makereplicate measurements of a sample. Also, the volume to be drawn intothe pipette tip should be larger than the volume marked on the tip toensure a uniform pull on the sample during a measurement. For a 30 μLvolume mark on the pipette tip, the micropipette was set to draw 42 μL.

Results

The instant example determined the ability to develop a higherconcentration liquid formulation of C1 INH as a monoformulation. Theinitial studies focused on concentration of the stock solution of C1 INHusing a spin concentration method. The solutions were initially adjustedfor pH but no other excipient was added. Three pH values wereinvestigated (pH 5.9, 6.9, and 7.9). Upon spin concentration, all of thesolutions remained clear up to concentrations up ˜500 U/ml(approximately 100 mg/ml) for all pH values tested (Table 1). While thesolubility limit was not reached in these studies, there were measurableincreases in viscosity as the concentrations exceeded 300 U/ml (FIG. 2).At all pH values, the viscosity begins to increase markedly when the C1INH concentration goes above 400 U/ml.

TABLE 1 Final concentrations (in U/mL) and viscosities for samplesprepared during the spin concentration experiments. These values werebased on the initial 160 U/mL concentration of the initial bulk drug.7.9 6.9 5.9 U/mL viscosity U/mL viscosity U/mL viscosity 93.12 0.99182.4 4.23 187.2 2.36 415.18 3.95 289.4 4.90 296.9 7.71 454.81 13.74378.6 12.08 396.7 5.46 501.17 30.43 479.0 14.67 478.8 24.09

A larger feasibility study was performed examining different buffers (20mM phosphate, 20 mM citrate, and 20 mM Tris) at each of the three targetpH values. Samples of both 400 U/ml and 500 U/ml were prepared andevaluated for stability after one week at 40° C. and after two weeks at25° C. The initial viscosity levels were well above the values for purewater (˜1 mPa-s), but well within the limits usually set for use as aninjectable product (Table 2). The viscosity values for the 400 U/mlsamples were less than at 500 U/ml, usually by 7 to 10 mPa-s. Uponstorage at 40° C. for one week, the viscosity of all of the samplesincreased. At pH 5.9, all of the same gelled, likely due to thermallyinduced aggregation. For the remaining formulations, the viscosityincreased to some degree. In some cases these values exceeded 30 mPa-s.The increase in viscosity was less upon 25° C. storage than at 40° C.There was little, if any change, for the samples at pH 6.9, indicatingthat pH 6.9 may be more favorable for long-term storage stability.

TABLE 2 Viscosity at t0 and after one week of storage at 40° C. (t1).Viscosity is reported in mPa-s. pH [C1 INH] Buffer t0 t1 t2 5.9 400phosphate 13.3 ± 0.6 gel 17.4 ± 2.1 500 24.6 ± 1.5 gel 36.9 ± 7.3 400histidine 14.7 ± 0.8 gel 19.1 ± 2.5 500 27.7 ± 3.8 gel 27.7 ± 3.8 6.9400 phosphate 12.2 ± 1.5 16.1 ± 0.6 11.9 ± 3.0 500 20.8 ± 2.0 35.3 ± 2.132.1 ± 7.7 400 citrate  7.4 ± 0.8  9.2 ± 0.7  7.1 ± 0.6 500 14.4 ± 3.219.8 ± 1.1 12.6 ± 0.5 7.9 400 phosphate  8.2 ± 1.2 12.8 ± 0.7 22.0 ± 3.5500 16.2 ± 1.4 23.1 ± 2.1 25.5 ± 7.5 400 tris 14.1 ± 0.7 18.7 ± 0.7 30.0± 3.8 500 20.5 ± 0.9 33.3 ± 6.2 31.0 ± 1.8

Notably, at pH 6.9, citrate formulations had lower viscosity values thanfor phosphate, while at pH 7.9, phosphate buffer produced lowerviscosities than tris buffer. Higher viscosities will mean greater forcewill be required to deliver a specified volume of the drug within acertain time frame.

The purity by RP HPLC was initially near 86 to 87% for the formulationsat pH 6.9 and above (Table 3). The initial levels were lower at pH 5.9,suggesting that some degradation had already occurred just in theprocess of preparing the samples. Upon storage for one week at 40° C.,the pH 5.9 samples gelled, making analysis by RP HPLC impossible. Forall of the other samples, the percent purity was essentially unchanged,indicating that little, if any, chemical degradation occurs for storageunder these conditions.

TABLE 3 Percent purity by RP HPLC upon storage at 25° C. (t2) or 40° C.(t1). pH [C1 INH] Buffer t0 t1 t2 5.9 400 phosphate 82.87 ± 0.75 gel81.10 ± 2.11 500 84.74 ± 1.24 gel 83.61 ± 1.02 400 histidine 84.11 ±1.53 gel 85.34 ± 1.55 500 86.36 ± 0.76 gel 82.99 ± 0.64 6.9 400phosphate 87.14 ± 0.67 88.59 ± 0.29 85.19 ± 2.00 500 86.44 ± 1.49 85.65± 1.32 84.07 ± 1.24 400 citrate 86.67 ± 1.36 82.92 ± 1.48 86.03 ± 0.87500 86.89 ± 1.24 86.74 ± 0.88 84.42 ± 1.19 7.9 400 phosphate 86.09 ±1.14 85.29 ± 0.84 85.98 ± 0.90 500 86.47 ± 1.15 83.57 ± 1.33 84.00 ±0.97 400 tris 87.14 ± 0.98 81.74 ± 7.89 86.14 ± 0.81 500 88.74 ± 0.8287.24 ± 1.47 87.30 ± 0.95

For samples stored for two weeks at 25° C., there were small losses,comparable to what was seen at t1. Together, the RP HPLC data indicatethat there are small losses due to chemical degradation. Higher pH seemsto diminish the rate of degradation and there may be some sensitivity tobuffer composition.

While the chemical stability of C1 INH seems to be unchanged uponstorage, there is come physical instability observed as indicated by SEC(Table 4). There are other proteins present in the C1 INH mixture,leading to an overall ‘purity’ of about ˜67% at t0. Upon storage at 40°C. for one week (t1), the overall monomer content of the samplesdecreased to 54-56% for the samples with pH 6.9 and higher. There waslittle difference between the two different pH conditions, the differentbuffers and the two protein concentrations. When stored for two weeks at25° C. (t2), the pH 5.9 samples did not gel, as they did at the higherstorage temperature. However, there was appreciably higher degradation,especially with histidine buffer. For these at pH 6.9 or 7.9, the lossas measured by SEC was about 2% or so, compared to the 10-12% loss atthe higher temperature for half of the time.

TABLE 4 Monomer content by SEC upon storage at 25° C. (t2) or 40° C.(t1). pH [C1 INH] Buffer t0 t1 t2 5.9 400 phosphate 68.32 ± 1.04 gel62.56 ± 0.94 500 67.19 ± 0.14 gel 61.46 ± 0.14 400 histidine 64.68 ±0.42 gel 46.58 ± 1.09 500 66.60 ± 0.08 gel 44.48 ± 1.04 6.9 400phosphate 67.85 ± 0.22 55.29 ± 0.36 500 67.41 ± 0.36 54.79 ± 0.14 65.45± 0.23 400 citrate 67.82 ± 0.07 56.14 ± 0.41 65.49 ± 0.16 500 67.43 ±0.30 56.59 ± 0.33 65.03 ± 0.36 7.9 400 phosphate 67.85 ± 0.09 54.96 ±0.52 61.31 ± 0.25 500 67.58 ± 0.40 55.57 ± 0.56 64.98 ± 0.50 400 tris67.63 ± 0.27 55.40 ± 0.30 65.70 ± 0.56 500 67.67 ± 0.47 56.18 ± 0.6466.19 ± 0.84

The data indicate that the rate of degradation will be about 13-fold to35-fold slower at 4° C. than at 25° C. The higher estimate comes fromusing an Arrhenius plot. The lower estimate comes from determine theaverage loss as the temperature is decreased by 5° C. and extrapolatingto a storage temperature of 4° C. Using the current data as anindicator, this predicts a loss of about 3 to 10% loss after two yearsat refrigerated temperatures. In other words, a liquid formulationappears to be quite stable based on these data. Furthermore, thedegradation rates are roughly comparable between the 400 U/mL and 500U/mL samples, suggesting that developing the higher concentrationformulation is just as viable.

The degradation rate is much faster at pH 5.9, leading to gelation at40° C. and greater losses at 25° C. Thus, further pH/buffer screeningwill focus on the pH 6.5 to 8.0 range. There is a clear buffer effect onviscosity and possibly also on stability.

The studies demonstrated that there is not a solubility limit topreparing C1 INH at concentrations up to 500 U/ml. There is an increasein viscosity once the concentrations reach the 400-500 U/ml range (whichis buffer dependent with citrate being better than phosphate which isbetter than Tris), but they are manageable and still allow faciledelivery by injection for standard syringe systems. In general, C1 INHis relatively stable to chemical degradation, as determined by RP HPLC.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. A method for treating hereditary angioedema(HAE), said method comprising subcutaneously administering to a subjectin need thereof a composition comprising a C1 esterase inhibitor, abuffer selected from citrate or phosphate, and having a pH ranging from6.5-8.0, wherein the C1 esterase inhibitor is administered at aconcentration of at least about 400 U/mL and a dose of at least about1000 U, and wherein the administration of the composition comprising theC1 esterase inhibitor increases the level of C1 esterase inhibitor inthe blood of the subject to at least about 0.4 U/mL, and wherein the C1esterase inhibitor comprises an amino acid sequence at least 95%identical to residues 23 to 500 of SEQ ID NO:1.
 2. The method of claim1, wherein the C1 esterase inhibitor is present in the composition in aconcentration of at least about 500 U/mL in the composition.
 3. Themethod of claim 1, wherein the composition is administered daily, everyother day, or every three days.
 4. The method of claim 1, wherein thecomposition is administered one, two, or three times a week.
 5. Themethod of claim 1, wherein the increased blood levels of C1 esteraseinhibitor are maintained at least 50% of the time betweenadministrations.
 6. The method of claim 1, wherein the HAE is Type I HAEor Type II HAE.
 7. The method of claim 1, wherein the administration ofthe composition comprising a C1 esterase inhibitor results in HAEprophylactic treatment.
 8. The method of claim 1, wherein theadministration of the composition comprising a C1 esterase inhibitorresults in treatment of an HAE attack.
 9. The method of claim 1, whereinthe administration of the composition results in at least a reduction inthe severity and/or number of HAE attacks.
 10. The method of claim 1,wherein the C1 esterase inhibitor is purified from plasma.
 11. Themethod of claim 1, wherein the C1 esterase inhibitor is recombinantlyproduced.
 12. The method of claim 1, wherein the C1 esterase inhibitorcomprises the amino acid sequence of residues 23 to 500 of SEQ ID NO:1.13. The method of claim 1, wherein the composition is a liquidformulation.
 14. The method of claim 1, wherein the composition isreconstituted from a lyophilized powder.
 15. The method of claim 1,wherein the administration of the composition comprising the C1 esteraseinhibitor increases the level of C1 esterase inhibitor in the blood ofthe subject up to about 1 U/mL.
 16. The method of claim 15, wherein theincreased blood levels of C1 esterase inhibitor are maintained at least50% of the time between administrations.
 17. The method of claim 1,wherein the buffer is citrate.
 18. The method of claim 1, wherein thebuffer is phosphate.